Glycosylation is the process of, or the result of, addition of carbohydrates (saccharides) to proteins and lipids. The glycosylation process is a co-translational and post-translational modification that takes place during the synthesis of membrane and secreted proteins. The majority of proteins synthesized in the rough endoplasmic reticulum (ER) undergo glycosylation (Brooks et al., Expert Rev Proteomics 3:345-59, 2006). Glycosylation is an enzyme-directed, site-specific process, having two specific types of attachment, N-linked glycosylation and O-linked glycosylation. N-linked carbohydrates are attached via N-acetylglucosamine linked to the amino acid asparagine at an amino acid consensus sequence “Asn-X-Ser/Thr.” The surrounding amino acids often dictate what type of, if any, glycosylation will take place. For example, if the middle amino acid in the consensus sequence is proline (Pro), no N-linked glycosylation takes place. Most O-linked carbohydrate covalent attachments to proteins involve a linkage between the monosaccharide N-acetylgalactosamine and the amino acids serine or threonine (Werner et al., Acta Pediatrica 96:17-22, 2007). There is no consensus sequence for O-linked glycosylation.
Glycosylation on protein may result in either addition of simple sugar residues such as mannose and glucose, or addition of more complex sugar residues such as sialic acid and fucose (Brooks et al., Expert Rev Proteomics 3:345-59, 2006), and branched chain sugars. Protein glycosylation serves several functions in vivo, including stabilization of the protein in the cytoplasm, increasing protein half-life, as well as regulating the activity of the protein or enzyme having the glycosyl residues (Werner et al., Acta Pediatrica 96:17-22, 2007). Thus, it is important to ensure that proteins express the correct glycosylation or the protein activity may be compromised or absent. The type of glycosylation on a protein often depends on the cell type in which the protein is synthesized, as well as the species of cell synthesizing the protein (Werner et al., supra; Brooks et al., supra). For example, bacteria and yeast do not synthesize complex glycans which are typically found on higher eukaryotic proteins (Brooks et al., supra). Even within mammalian species (e.g., human and hamster), and from tumor cells to normal, non-malignant cells, glycosylation patterns can be different (Werner et al., supra). Thus, the type of cell system in which the protein is produced has a significant influence on the resulting glycosylated product (Werner et al., supra).
Recombinantly produced proteins have provided a significant improvement to the study of proteins in both clinical and research settings. The large scale production of recombinant proteins has enabled the study of protein activity in vitro, and recombinant proteins have recently been used as therapeutic agents in the clinical setting. For example, recombinant interleukin-2 has been administered to cancer patients to boost the immune system after chemotherapy, and recombinant growth factors, such as human growth hormone, erythropoietin and granulocyte colony stimulating factor, and blood factors, such as Factor VIII and Factor VII, are used in the treatment of various disorders.
Although recombinant proteins provide an advantage as therapeutic proteins, they also exhibit certain drawbacks. It can be difficult to produce sufficient amounts of recombinant protein for therapeutic use in human cells in a cost efficient manner, and recombinant protein made in such cells as Escherichia coli and other bacteria do not necessarily fold properly, are not glycosylated, and/or must be manipulated once isolated to manufacture proteins in a form active in the human body. Additionally, glycosylation of proteins in human cells is often more complex than that seen in commonly used protein expression systems, such as bacteria, insects and even higher mammals. For example, insect cells such as Spodoptera rarely generate proteins having higher order sugar structure of the types produced in mammals (Altmann et al., Glycoconjugate J 16:109-123, 1999). Further, although most mammals express higher order sugars comprising such structures as fucose and sialic acid residues, these sugar moieties may not be chemically attached in the same manner as the sugars in proteins produced in human cells (Jenkins et al., Nature Biotechnology 14:975-981, 1996; U.S. Pat. No. 5,047,335).
Administration of therapeutic proteins is often used in order to correct a deficiency or functional defect in the endogenously expressed protein. Recombinant insulin and insulin analogs (Vajo et al., Pharmacol Rev. 52:1-9, 2000) are administered to diabetic patients to make up for the lack of naturally produced insulin Recombinant Factor VIII and sequence analogs of Factor VIII are administered to patients suffering from hemophilia A to correct a deficiency in Factor VIII levels resulting in aberrant blood clotting (Gruppo et al., Haemophilia. 9:251-60, 2003). In recombinant therapies of these types, it is useful to determine the levels of recombinant protein in serum or other sample in order to determine the half-life of the drug and other pharmacokinetics, such as absorption, in the patient. However, it can be difficult to determine the difference between the endogenous protein and the recombinant protein, since they are essentially the same protein.
Thus, there remains a need in the art to develop methods to differentiate the amount of naturally-derived protein from the exogenous protein in a sample and to detect the levels of endogenous and exogenous recombinant protein administered to a patient in order that treatment regimens may be optimized.